We have investigated the influence of insulin on the activity of casein kinase II (CKII) in skeletal muscle of 7 insulin-sensitive, 4 insulin-resistant, non-diabetic, and 5 diabetic Pima Indians during a 2-hour hyperinsulinemic, euglycemic clamp. In sensitive subjects, CKII was transiently activated (42%) reaching a maximum over basal activity at 45 minutes before declining. CKII was also stimulated in resistant subjects (19%) and diabetics (34%), but the activity remained elevated throughout the clamp. The magnitude of activation of CKII by insulin in muscle of resistant subjects may have been limited by the fact that basal CKII activity in this group was 40% higher than in either sensitive or diabetic subjects. The higher basal CKII activity in resistant subjects was not explained by a higher concentration of CKII, protein as determined by Western blotting. Among the three groups, basal CKII activity was correlated with fasting plasma insulin concentrations suggesting that the higher basal activity in resistant subjects resulted from their higher plasma insulin concentrations. Extracts of muscle obtained from all three groups either before or after insulin administration were treated with immobilized alkaline phosphatase, which reduced and equalized CKII activity in the extracts. These results suggest that insulin stimulates CKII activity in human skeletal muscle by a mechanism involving phosphorylation of either CKII or of an effector molecule and support the idea that elevated basal activity in resistant subjects results from insulin action. Our studies suggest that the ability of insulin to activate CKII in skeletal muscle is not impaired in insulin-resistant Pima Indians, and that the biochemical lesion responsible for insulin resistance occurs either downstream from CKII or in a different pathway of insulin action.